To investigate the effect of proteasomes reactivator REG gamma on cell cycle and apoptosis in vitro and in vivo. In vitro, we first constructed recombinant plasmid of PcDNA3.1-REGgamma and then transfected REGgamma into MDA-MB-231 cell line. We confirmed the transfection efficiency by Western blot. Subsequently, we observed cell growth, cycle ...

Gene transfer methods for producing recombinant cell lines are often not very efficient. One reason is that the recombinant DNA is delivered into the cell cytoplasm and only a small fraction reaches the nucleus. This chapter describes a method for microinjecting DNA directly into the nucleus. Direct injection has several ...

Inhibitor of differentiation 2 (Id2) is a critical factor for cell proliferation and differentiation in normal vertebrate development. Most of the biological function of Id2 has been ascribed to its helix-loop-helix motif. Overexpression of Id2 is frequently observed in various human tumors, but its role for invasion potential in tumor ...

A major obstacle in studying monocyte cell biology has been the resistance of these cells to genetic manipulation, particularly when using nonviral methods. In the approach outlined in this chapter, we describe a strategy for stable gene silencing of monocytic cells. Using a vesicular stomatitis virus (VSV)-pseudotyped lentiviral vector expressing ...

The transfection of expression constructs encoding a variety of transgenes is a widely used method to study gene function in cultured cells. Especially when the efficiency of the knock-down of target proteins via small interfering RNAs (siRNAs) is to be determined by quantitative Western blotting, large proportions of untransfected cells ...

In this chapter, protocols for the growth and transfection of Human Embryonic Kidney (HEK) 293T cells for small scale expression screening and large scale protein production are described. Transient expression in mammalian cells offers a method of rapidly producing glycoproteins with a relatively high throughput. HEK 293T cells, in particular, ...

BACKGROUND AND OBJECTIVE: Leucine-rich repeats and immunoglobin-like domains 3 (LRIG3), a member of LRIG gene family, is down-regulated in various human cancers, but its functions are still unclear. This study was to explore the effect of RNA interference (RNAi)-mediated LRIG3 gene silencing on the proliferation of glioma GL15 cells and ...

BACKGROUND: Hepatocellular carcinoma (HCC) is a lethal malignancy with high rate of metastasis and poor prognosis. There are no effective managements to block metastasis of HCC. Programmed cell death 4 (PDCD4) is found to be a tumor transformation suppressor. Among investigations on effects of PDCD4, little is about the metastatic ...

BACKGROUND: Fully differentiated adipocytes are considered to be refractory to introduction of siRNA via lipid-based transfection. However, large scale siRNA-based loss-of-function screening of adipocytes using either electroporation or virally-mediated transfection approaches can be prohibitively complex and expensive. METHODOLOGY/PRINCIPAL FINDINGS: We present a method for introducing small interfering RNA (siRNA) into ...

To investigate the pro-angiogenic factors stimulated by human papillomavirus type 16 E6 and E7 protein in C33A and human fibroblasts. HPV-16 E6 and E7 genes were transfected into C33A and HFB to detect the profiling of angiogenesis-associated factors with the TranSignal angiogenesis antibody array. The mRNA and protein levels of ...

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be ...

Microarrays have become common tools for approaching different experimental questions: DNA, protein and peptide arrays offer the power of multiplexing the assay and by means of miniaturization technology, the possibility to reduce cost and amount of samples and reagents. Recently, a novel technology for functional assays has been proposed. Sabatini ...

Ribonucleotide reductase (RR) has an essential role in DNA synthesis and repair and is a therapeutic target in a number of different cancers. Previous studies have shown that RNAi-mediated knockdown of either the RRM1 or RRM2 subunit sensitizes cells to the cytotoxic effects of the nucleoside analogs and more recently ...

Salivary adenoid cystic carcinoma (SACC) is one of the most common malignancies of salivary gland. Recurrence or/and early metastasis is its biological properties. In SACC, neoplastic myoepithelial cells secrete proteoglycans unconventionally full of the cribriform or tubular and glandular structures of SACC. Literatures have demonstrated that extracellular matrix provided an ...

An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, ...

The aim of this work is to investigate the effect of betaine substitution degree of chitosan N-betainates (CsB) on cellular uptake, cytotoxicity and transfection efficiency of CsB/DNA complex nanoparticles (CsBNs) against COS-7 and MDA-MB-468 cells. The polymers with three substitution degrees (CsB12, CsB47 and CsB85) complexed with pDNA formed CsBN12s, ...

The production of transgenic pigs using somatic cell nuclear transfer (scNT) has been widely described, but a technique for removing nontransfected donor cells and for creating different founder animals has not yet been fully elucidated. In this study, four different expression vectors (pBC1hEPO, pMARBC1hEPO, pBC1hEPOwpre and pMARBC1hEPOwpre) were compared to ...

Many cancers develop different means of escaping destruction by the immune system, such as resistance to Fas ligand (FasL)-Fas interaction-mediated apoptotic signals. Decoy receptor 3 (DcR3), a soluble receptor for FasL, is highly expressed in cancer cells and plays a significant role in immune suppression and tumor progression. However, how ...

We report the transfection of human hepatocarcinoma (HepG2) cells by femtosecond (fs) laser pulses at 1554 nm. It was found that HepG2 cells could be perforated transiently to admit propidium iodide by that laser. This photoporation was safe, as the membrane resealed itself within a short time and no mitochondrial ...

Carbonic anhydrase III (CAIII) is distinguished from the other members of the CA family by low carbon dioxide hydratase activity, resistance to the CA inhibitor acetazolamide, and a predominant expression in the liver of males. In this report the effects of CAIII expression on liver cancer cells invasiveness were explored. ...

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the ...

Electroporation is a valuable tool for nucleic acid delivery because it can be used for a wide variety of cell types. Many scientists are shifting toward the use of cell types that are more relevant to in vivo applications, including primary cells, which are considered difficult to transfect. The ability ...

The induction of therapeutic angiogenesis with gene therapy approaches has received considerable interest and some limited clinical success. A major drawback to this approach is a lack of understanding of the pharmacokinetics of therapeutic protein delivery. This has become increasingly more relevant as recent studies have illustrated a defined therapeutic ...

RNA interference is a powerful tool for the functional analysis of proteins by specific gene knockdown. In this study, we devised a rapid and efficient way to screen suitable siRNA sequences and subsequently employ them for specific gene knockdown in usually hard-to-transfect lymphoid cell lines, using a self-inactivating lentiviral vector. ...

Kang Hyunmin H Division of Molecular Pharmaceutics, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, - - 2008

Targeted delivery can potentially improve the pharmacological effects of antisense and siRNA oligonucleotides. Here, we describe a novel bioconjugation approach to the delivery of splice-shifting antisense oligonucleotides (SSOs). The SSOs are linked to albumin via reversible S-S bonds. The albumin is also conjugated with poly(ethylene glycol) (PEG) chains that terminate ...

The use of RNA interference (RNAi) to deplete individual proteins from cells or tissue has revolutionised our ability to characterise gene function. The placenta is an attractive target for studies in which the role of specific proteins can be compared with cell culture models and explanted villous tissue where physiological ...

Colorectal carcinoma (CRC) is often lethal when invasion and/or metastasis occur. NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), an enzyme involved in prostaglandin (including PGE(2)) bio-inactivation, is down-expressed in several epithelial malignancies including CRC. Although its role in the suppression of colon tumorigenesis has been well learned, little is known about the role ...

BACKGROUND: Cell-penetrating peptides (CPPs) could potentially be used as vectors for intracellular delivery of proteins, peptides and nucleic acids. The present study examined different CPPs, such as TAT-derived and arginine rich sequences, as well as model amphiphilic peptide, with respect to transfection efficiency of pegylated polyethylenimine (PEI) in A549, Calu-3 ...

Human mesenchymal stem cells-like cells (hMSCs-like cells) were used as a tumor treatment platform for the systemic delivery of immunotoxin genes. VEGF165-PE38 recombinant immunotoxin served as the model system. hMSCs-like cells were isolated, expanded, and electroporated with the pIRES2-VEGF165PE38-EGFP plasmid. RT-PCR and ELISA were used to confirm the expression of ...

Although glucocorticoid (GC) has been reported to inhibit macrophage killing activity and cytokine production in response to proinflammatory stimuli, the effect of GC on macrophage proliferation is controversial. In our previous study, we found that inhibition of glucocorticoid receptor (GR) expression in murine macrophage cell line RAW264.7 cells (RAW-GR(-) cells) ...

Primary cell cultures from crustacea have been initiated since the 1960s, yet no permanent cell line is available. Primary cells have a limited proliferative capacity in culture due to cellular senescence, which is regulated by a group of dominant senescence genes. The aim of this research was to manipulate cell ...

GFP plasmids were constructed and used for transfection of blood and sporozoite stages of P. vivax. GFP transformed parasites were observed when transfection was performed using blood stage parasites. Transfection of GFP plasmids into liver cells could enhance plasmid uptake of the sporozoites after invasion of the liver cells. Optimization ...

The effect of target-directed regulation of the uncoupling protein-2 (UCP-2) gene expression on the ischemia-reperfusion injury of hepatocytes under different conditions was investigated. The expression plasmid and RNAi plasmid targeting UCP-2 gene were constructed and transfected into normal hepatocytes and fatty liver cells, respectively. The expression of UCP-2 mRNA was ...

Lipoplexes and polyplexes, electrostatic complexes between a plasmid DNA and cationic lipids or polymers are chemical systems that are developed for gene delivery. Considerable efforts have been done to delineate the exact knowledge of their entry mechanisms and the intracellular routing of the plasmid DNA that are of major importance ...

Polyethylenimine (PEI) is an efficient vector for in vitro and in vivo gene transfer into respiratory cells. Glycosylated PEIs were shown to enhance in vitro gene transfer by favoring the complex entry into the airway cells. The aim of our study was to evaluate the in vivo efficiency of gene ...

OBJECTIVE: To investigate the relationship between the S100P and the sensitivity of ovarian cancer to chemotherapeutics. METHOD: We established stable cell lines of ovarian cancer cells, SKOV3 and OVCAR3, that overexpress human S100P. We also transiently transfected the parent cell lines with S100P-targeted siRNA for down-regulation of S100P expression. The ...

A white ear lobe chicken embryo (WELCE) fibroblast cell bank, containing 322 tubes of frozen cells, was successfully established from primary explants of 57 embryo samples. The cells were morphologically consistent with fibroblasts, and the growth curve was sigmoidal with a population doubling time (PDT) of 48 h. Karyotyping and ...

Cell migration plays a major role in a variety of biological processes and a detailed understanding of associated mechanisms should lead to advances in the medical sciences, for example, in drug discovery for cancer therapy. However, the traditional methods used for analysis of cell migration cannot easily be scaled up ...

Caveolin-1 (CAV1) is an integral membrane protein that may function as a scaffold for plasma membrane proteins and acts as a tumor suppressor protein. One causative factor of chemotherapy-resistant cancers is P-plycoprotein (P-gp), the product of the multidrug resistance-1 gene (MDR1), which is localized in the caveolar structure. Currently, the ...